Establishment and application of a multiplex PCR assay for detection of Rhizoctonia cerealis, Bipolaris sorokiniana, and Fusariumspp. in winter wheat

Rhizoctonia cerealis, Bipolaris sorokiniana, and Fusariumspp. are three wheat-damaging soil-borne fungal pathogens causing sharp eyespot, common root rot, and crown rot, respectively. This study aimed to develop a multiplex PCR (mPCR) system to simultaneously detect these fungal pathogens. The mPCR assay was performed on wheat seedlings collected from a field in the Shandong Province. Specific primers were designed on the basis of the sequences of following genes: internal transcribed spacer (ITS) of R. cerealis, ITSof B. sorokiniana, and translation elongation factor (TEF1-α) of Fusariumspp., generating amplicons of 174, 418, and 600 bp, respectively. The mPCR system was optimized through an orthogonal design L18 (37) (three levels of five factors: three sets of primers, TaqDNA polymerase, and dNTPs) and statistical analysis. No nonspecific products were obtained upon amplification of nontarget species of the fungi. Sensitivity analysis revealed that the limit of detection of the mPCR assay for each pathogen was 100 pg. The mPCR results were further confirmed via subsequent visual diagnosis. The system developed herein provides a rapid, effective, and valuable method to simultaneously detect three soil-borne pathogens, thus facilitating prompt treatment and prevention of root diseases of wheat.